Synthesis and Pharmacological Characterization of Visabron, a Backbone Cyclic Peptide Dual Antagonist of α4β1 (VLA-4)/α9β1 Integrin for Therapy of Multiple Sclerosis

Integrins α4β1/ α9β1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4β1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought. In the present study, we synthesized and characterized visabron c (4–4), a backbone cyclic octapeptide based on the sequence TMLD, a non-RGD unique α4β1 integrin recognition sequence motif derived from visabres, a proteinous disintegrin from the viper venom. Visabron c (4–4) was selected from a minilibrary with conformational diversity based on its potency and selectivity in functional adhesion cellular assays. Visabron c (4–4)’s serum stability, pharmacokinetics, and therapeutic effects following ip injection were assessed in an experimental autoimmune encephalomyelitis (EAE) animal model. Furthermore, visabron c (4–4)’s lack of toxic effects in mice was verified by blood analysis, tissue pathology, immunogenicity, and “off-target” effects, indicating its significant tolerability and lack of immunogenicity. Visabron c (4–4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4–4) as a safe alternative peptidomimetic lead compound/drug to monoclonal anti-α4 integrin antibodies, steroids, and other immunosuppressant drugs. Moreover, visabron c (4–4) design may pave the way for developing new therapies for a variety of other inflammatory and/or autoimmune diseases.


MNSANNPCCDPKTCKPRKGEHCVSGPCCRNCKFLLPGTICKRTMLDGLNDYCTG V(I/T)(T/S)(P/D)DCPRNPW(Y/K)SEEED
a Visabres molecule was reduced and cysteines were ethylpyridylated and the amino acid sequence of the subunits was established by a combination of mass spectrometry and Nterminal sequencing of its proteolytic fragments. Cysteine used for sequence alignment is underlined; TMLD a4-integrin binding motif is in italics; Amino acids with ambiguity are in parenthesis.
b B subunit sequence is tentative and needs to be completed and confirmed by DNA sequencing. MALDI-TOF mass spectroscopic analysis of intact Visabres yielded an apparent molecular weight of 16,850 Daltons, whereas subunit A and B indicated an apparent molecular weight of 8,160 and 8743 Daltons, each containing 10 ethylpyridylated cysteine residues, respectively.              in the blood that is eliminated rapidly while the pharmacology activity kinetics differ significantly (indirect PD).    ***In three mice, it was found in the last part of the colon, rectum and the adjacent anal unhaired skin a severe purulent, necrotizing inflammation. This finding was most probably not related to the tested compound and was considered as an accidental finding since was also found in a few control animals (data not shown), probably due to allergic food sensitivity.

Supplemental Methods
Phospholipase A2: Activity was determined by a commercial colorimetric assay purchased from Abcam Co. (Cambridge, UK) that provides an accurate and convenient method for measurement of secretory PLA2 (sPLA2) activity. This assay uses the 1, 2-dithio analog of diheptanoyl phosphatidylcholine which serves as a substrate for most PLA2s (e.g., bee and cobra venoms, pancreatic, etc.). Upon hydrolysis of the thio ester bond at the sn-2 position by PLA2, free thiols were detected using DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid) (www.abcam.com/ab133089). Substrates incubated without enzyme were used as controls. After 24 h of incubation, 20 μl of each preparation was mixed with electrophoresis sample buffer and the proteolytic reaction products were evaluated by SDS-PAGE. In order to determine the nature of the protease activity different protease inhibitors were used, namely: EDTA (10 mM), o-phenantroline (100 μM), leupeptin (10 μM). All the inhibitors were obtained from Sigma (Sigma Chemical Co., St Louis, MO).
Hemorrhagic activity: was assessed as described Theakston and Reid 3 . Venom and FPLC fractions at doses of 10 to 50 µg in 50 µL of saline solution were injected by intradermal route in BALB/c male mice. Hemorrhagic measurements were recorded after two hours. Diameters of hemorrhagic area were measured, and the minimum hemorrhagic dose (MHD) was defined as the venom dose that induced a lesion of 10 mm in diameter.
Sequence characterization of ethylpyridylated Visabres subunits: 1 mg/mL Visabres was solubilized in in 0.1 M Tris-HCl, pH 8.5, 4 mM EDTA, 6 M guanidine hydrochloride and reduced with 3.2 mM dithiothreitol for 3 h at room temperature in the dark. Reduced proteins were alkylated by addition of a 2-fold molar excess of 4-vinylpyridine over the reducing reagent. Ethylpyridylated (EP) A and B subunits were isolated by reversed-phase HPLC on a C-18 column. The isolated EPsubunits were submitted for N-terminal sequencing using an Applied Biosystems 477A and MALDI-TOF mass spectrometry using a PE-Biosystems Voyager-DE Pro instrument. The primary structures of EP-polypeptides was deduced from the N-terminal sequence analysis of overlapping peptides, obtained by proteolytic digestions with trypsin (Sigma), endoproteinase Lys-C (Boehringer Mannheim), and endoproteinase Asp-N (Boehringer Mannheim) (2 mg/mL protein in 100 mM ammonium bicarbonate, pH 8.3, for 24 h at 37 °C using an enzyme: substrate ratio of 1:100 (w/w) and degradation with CNBr (10 mg/mL protein and 100 mg/mL CNBr in 70% (v/v) formic acid for 6 h at room temperature, under N2 atmosphere and in the dark. Peptides were separated by reverse-phase HPLC, using a 4 × 250 mm C18 (5 µm particle size) Lichrospher RP100 (Merck) column eluting at 1 mL/min with a linear gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B). Visabres subunits were collected manually and dried in a SpeedVac (Savant). The purity and molecular mass of the reverse-phase-isolated proteins were checked by SDS/14% PAGE, Nterminal sequencing (using an Applied Biosystems Procise 492 sequencer), MALDI-TOF (matrixassisted laser-desorption ionization-time-of-flight) MS using a Voyager-DE Pro instrument (Applied Biosystems), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Sigma) saturated in 70% acetonitrile and 0.1% TFA as matrix, and electrospray ionization MS with a triple quadrupole-ion trap hybrid instrument (QTrap from Applied Biosystems) equipped with a Nano spray source (Protana). For peptide sequencing, the protein digest mixture was subjected to electrospray ionization MS/MS analysis using a QTrap mass spectrometer, equipped with a nanospray source. Doubly charged ions selected after Enhanced Resolution MS analysis were fragmented using the Enhanced Product Ion with Q0 trapping option at 250 atomic mass units/s (a.m.u./s ) across the entire mass range. For MS/MS experiments, Q1 was operated at unit resolution, the Q1-Q2 collision energy was set to 35 eV, the Q3 entry barrier was 8 V, the LIT (linear ion trap) Q3 fill time was 250 ms, and the scan rate in Q3 was 1000 a.m.u./s. CID spectra were interpreted manually or using the online form of the MASCOT program (http://www.matrixscience.com). Quantitation of free cysteine residues and disulfide bonds was done as previously described 4 .

Pharmacokinetics of Visabron c (4-4) following iv injection:
Animals-Male Wistar rats (275−300 g, purchased from Harlan, Israel), were used for the surgical procedure. An indwelling cannula was implanted into the right jugular vein, and exteriotrated at the dorsal neck to enable serial systemic blood sampling. After completion of the surgical procedure, the animals were transferred to cages to recover overnight (12−18 h). The animals had free access to water and food during the recovery period and throughout the entire experiment. All surgical and experimental procedures were approved by the Animal Experimental Ethics Committee of the Hebrew University, Hadassah Medical School, Jerusalem.
Plasma Stability Studies-Visabron c(4-4) (10 μg/mL) was mixed with fresh plasma from male Wistar rats (Harlan, Israel) and incubated at 37 °C for 180 min. Triplicate samples were taken at time 0 and after 5, 10, 15, 30, and 60 and 90 min . Samples (50 μL) were withdrawn and the reaction was terminated by adding 100 μL of ice cold acetonitrile (ACN). Each sample was spiked with metoprolol (IS, 5 μg/mL) and centrifuged (14635 g, 10 min). The supernatant was transferred to fresh glass tubes and evaporated to dryness. The glass tubes were reconstituted in 80 μL of mobile phase and centrifuged a second time (14635 g, 10 min).The reactions were analyzed using HPLC-MS.
PK Study-A single dose of Visabron c  in sterile water (300 μL of a 0.514 mg/mL solution, which was equivalent to a final dose of 0.514 mg/kg) was administered i.v. via the indwelling jugular cannula to each rat. Following dose administration, blood samples were taken at pre-dose, 10, 15, 30, 60, 90 and 130-min from the onset of the drug administration (time zero) via the right jugular vein. Approximately 350 μL of whole blood was removed, and placed in sodium-heparin containing tubes, and immediately placed in wet ice. Blood was centrifuged at 5322 g for 10 min and a 150 μL aliquot of plasma was transferred to fresh centrifuge tubes and stored at −20 °C pending analysis. PK analysis was performed using standard non-compartmental analysis models in Phoenix WinNonlin®.

Bioanalytical Method for the Detection of Vis-c(4-4) in Rat Plasma:
Plasma samples (150 μL) were spiked with metoprolol (20 μL, 10 μg/mL) as an internal standard. 200 μL of ACN was added to each sample and vortex-mixed for 1 min. The samples were then centrifuged (14635 g, 10 min), and the supernatant was transferred to fresh glass tubes and evaporated to dryness (Vacuum Evaporation System, Labconco, Kansas City, MO, USA). The glass tubes were reconstituted in 80 μL of mobile phase and centrifuged a second time (14635 g, 10 min). The amount of the compounds was determined using an HPLC−MS Waters 2695 Separation Module, equipped with a Micromass ZQ detector. The resulting solution was injected (10 μL) into the HPLC system. The system was conditioned as follows : Xterra MS C18, 3.5 µm, 100A, 100 x 2.1 mm column (Waters), an isocratic mobile phase of ACN : water supplemented with 0.1% formic acid (15:85, v/v) and a flow rate of 0.2 mL/ min at 35 °C was used. The limit of quantification the peptide was 25 ng/mL. Calibration curve was prepared by spiking the standard working stock solution with 15 μL of the different concentrations, 20 μL IS (10 μg/ mL) and 135 μl blank rat plasma in a centrifuge tube on the analysis day. The plasma calibration curve had a range of 25 ng/Ml to 2.5 μg/mL.

Blood cell counting and biochemistry analyzes:
Male C57BL/6 mice were injected intravenously with 0.2 ml of peptide in a dose of 500 mg/kg. No immediate toxicity symptoms were measured and thereafter, acute tolerability was observed. Since the hematopoietic system is one of the most sensitive parameters to assess the toxicity of drugs in humans and animals, tail vein blood samples were taken from control (n=3) and 250 mg/kg Visabron c (4-4) injected mice (n=5), after 24 hours from injection and submitted for hematocrit cell counting and biochemical analysis. No significant differences in blood count and electrolytes of special importance are normal liver (ALP, alkaline phosphatase; ALT, alanine transaminase and AST, aspartate transaminase) functions and normal kidney (creatinine and blood urea nitrogen) functions, indicating sufficient organ integrity at the end of the experimental period. The data obtained was similar to hematological parameters reported for this mice strain 5 , and clearly indicates lack of differences on kidney and liver parameters between the two mice groups.

Visabron c (4-4) safety by in vitro evaluation using PhosphoSens-CSox based kinase assays:
A powerful method was used to measure the activity of recombinant protein kinases using a homogeneous and continuous (kinetic) format, where the level of chelation-enhanced fluorescence (CHEF) that is directly proportional to the amount of phosphorylated, real-time sensors consisting of sulfonamido-oxine (Sox) chromophore, linked to a peptide or protein substrates of selective kinases 6 . This assay is ideal for elucidating drug mechanism of action and is increasingly being applied earlier in the drug development workflow, to address ''off target'' effects and/or the challenges and opportunities for next generation lymphocyte protein kinase inhibitors.
Kinase activity measurements: Kinase activity was measured continuously using the PhosphoSens® technology (AssayQuant Technologies Inc., Marlborough, MA) according to the manufacturers' recommendations. This one-step homogeneous assay format uses chelation-enhanced fluorescence via optimized substrate sensors containing the unnatural fluorogenic amino-acid Sox.